Cells within many tissues and organs are continually replenished throughout our lives. This is accomplished by stem cells, which balance self-renewal with more differentiated cell types. The mechanisms that control this balance are not clear. In tissues maintained by stem cells, the differentiating daughters undergo mitotic expansion before generating tissue-specific cell types. The mechanisms that control transit amplification are also not clear. Both of these phenomena are featured in Drosophila spermatogenesis, where stem cell daughters choose between self-renewal and differentiation, and where transit amplifying gonial cells switch to spermatocyte development. Gonial cells divide four times; the counting and effector mechanisms regulating this are unknown. Aim 1 tests the hypothesis that germ cells count intrinsically, and then collaborate with surrounding somatic cells to coordinate the spermatocyte transition. We discovered that a somatic cell signal promotes differentiation of stem cell daughters, while other labs discovered a signal that promotes self-renewal. Aim 2 tests the hypothesis that self-renewal and differentiation are indeed balanced by these competing signals, and then investigate how they compete. To generate and begin to test biologically-based hypotheses for other candidate factors and signaling pathways that legislate between renewal and differentiation we have conducted transcript profiling analyses of self-renewing cells or their differentiating daughters. Aim 3 proposes to complete these studies and conduct functional tests on selected candidates.